Methods for isolating microvesicles and extracting nucleic acids from biological samples

ABSTRACT

The invention provides novel methods and kits for isolating nucleic acids from biological samples, including cell-free DNA and/or cell-free DNA and nucleic acids including at least RNA from microvesicles, and for extracting nucleic acids from the microvesicles and/or from the biological samples.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/325,021, filed Jan. 9, 2017. U.S. patent application Ser. No.15/325,021 is a national stage application, filed under 35 U.S.C § 371,of PCT Application No. PCT/US2015/039760, filed Jul. 9, 2015, whichclaims priority to and the benefit of U.S. Provisional Application No.62/022,538, filed Jul. 9, 2014, U.S. Provisional Application No.62/079,763, filed Nov. 14, 2014, and U.S. Provisional Application No.62/166,890, filed May 27, 2015. The contents of each of theaforementioned patent applications are incorporated herein by referencein their entireties.

FIELD OF THE INVENTION

The invention provides novel methods and kits for isolating nucleicacids from biological samples, including cell-free DNA and/or cell-freeDNA and nucleic acids including at least RNA from microvesicles, and forextracting nucleic acids from the microvesicles and/or from thebiological samples.

BACKGROUND

Membrane vesicles that are shed by cells are referred collectively asmicrovesicles. Microvesicles from various cell sources have beenextensively studied with respect to protein and lipid content. Recently,microvesicles have been found to also contain both DNA and RNA,including genomic DNA, cDNA, mitochondrial DNA, microRNA (miRNA), andmessenger RNA (mRNA).

Due to the genetic and proteomic information contained in microvesiclesshed by cells, current research is directed at utilizing microvesiclesto gain further insight into the status of these cells, for example,disease state or predisposition for a disease. In addition, currentresearch is also directed at utilizing cell-free DNA to gain furtherinsight into the status of cells.

Accordingly, there is a need for methods of isolating cell-free DNA andfor isolating microvesicles from biological samples and methods ofextracting high quality nucleic acids for accurate diagnosis of medicalconditions and diseases.

SUMMARY OF THE INVENTION

The present invention provides methods for isolation of cell-free DNA(“cfDNA,” also known as circulating DNA) and/or for the combinedisolation of cfDNA and nucleic acids including at least the RNA frommicrovesicles from a sample by capturing the DNA, DNA and RNA, and/ormicrovesicles to a surface, subsequently lysing the microvesicles torelease the nucleic acids, particularly RNA, contained therein, andeluting the DNA and/or DNA and nucleic acids including at least RNA fromthe capture surface. Those of ordinary skill in the art will appreciatethat the microvesicle fraction also includes DNA. Thus, lysis of themicrovesicle fraction releases both RNA and DNA. Furthermore, the DNAisolated can be from any of a variety of sources including, but notlimited to nucleosomes and other cell-free DNA sources.

Previous procedures used to isolate and extract nucleic acids from asample, e.g., cfDNA and/or DNA and nucleic acids including at least RNAfrom the microvesicle fraction of a sample, relied on the use ofultracentrifugation, e.g., spinning at more than 10,000×g for 1-3 hrs,followed by removal of the supernatant, washing the pellet, lysing thepellet and purifying the nucleic acids, e.g., DNA and/or DNA and RNA ona column. These previous methods demonstrated several disadvantages suchas being slow, tedious, subject to variability between batches, and notsuited for scalability. The methods and kits for isolation andextraction provided herein overcome these disadvantages and provide aspin-based column for isolation and extraction that is fast, robust andeasily scalable to large volumes.

The methods and kits isolate and extract nucleic acids, e.g., DNA and/orDNA and nucleic acids including at least RNA from a sample using thefollowing general procedure, which is referred to herein as “EXO52.”First, the nucleic acids in the sample, e.g., the DNA and/or the DNA andthe microvesicle fraction, are bound to a capture surface such as amembrane filter, and the capture surface is washed. Then, a reagent isused to perform on-membrane lysis and release of the nucleic acids,e.g., DNA and/or DNA and RNA. Chloroform extraction is then performedusing PLG tubes, followed by ethanol conditioning. The nucleic acids,e.g., DNA and/or DNA and RNA, are then bound to a silica column, washedand eluted.

The membranes used in the EXO52 methods and kits have large pores andare positively charged. In some embodiments, more than one membrane isused in the EXO52 methods and kits, for example, two or more membranesare used. In some embodiments, three membranes are used. The number ofmembranes used in the EXO52 methods and kits correlates with the totalvolume of sample that can be analyzed at one time. In some embodiments,about 1 ml of samples is processed for each layer of membrane used inthe EXO52 methods and kits.

In some embodiments, the membrane is a positively charged membrane. Insome embodiments, the capture surface is an anion exchanger. In someembodiments, the capture surface is an anion exchanger with quaternaryamines. In some embodiments, the capture surface is a Q membrane, whichis a positively charged membrane and is an anion exchanger withquaternary amines. For example, the Q membrane is functionalized withquaternary ammonium, R—CH₂—N⁺(CH₃)₃. In some embodiments, the membranehas a pore size that is at least 3 μm.

Purification of the sample, including the microvesicle fraction, isperformed using ion exchange techniques. In some embodiments, the ionexchange technique is a technique selected from those shown in theworking examples provided herein.

In some embodiments, the agent used for on-membrane lysis is aphenol-based reagent. In some embodiments, the lysis reagent is aguanidinium-based reagent. In some embodiments, the lysis reagent is ahigh salt based buffer. In some embodiments, the lysis reagent isQIAzol. In some embodiments, the lysis reagent is a phenol-based lysisreagent, e.g., QIAzol, and it is used at a volume of about 700 ul.

In one aspect, the method for extracting nucleic acids from a biologicalsample comprises (a) providing a biological sample; (b) contacting thebiological sample with a capture surface under conditions sufficient toretain the microvesicle fraction on or in the capture surface; (c)lysing the microvesicle fraction while the microvesicles are on or inthe capture surface; and (d) extracting the nucleic acids from themicrovesicle fraction. Alternatively, the method for extracting nucleicacids from the biological sample further comprises eluting themicrovesicle fraction from the capture surface after step (b),collecting the eluted microvesicle fraction, and extracting the nucleicacids from the eluted microvesicle fraction. Optionally, the elutedmicrovesicle fraction can be concentrated by a spin concentrator toobtain a concentrated microvesicle fraction, and the nucleic acids aresubsequently extracted from the concentrated microvesicle fraction.

In another aspect, the method for extracting nucleic acids from abiological sample comprises (a) providing a biological sample; (b)contacting the biological sample with a capture surface under conditionssufficient to retain the microvesicle fraction on or in the capturesurface; and (c) eluting the microvesicle fraction while themicrovesicles are on or in the capture surface. The eluted microvesiclefraction can then be processed for further analysis. Optionally, theeluted microvesicle fraction can be concentrated by a spin concentratorto obtain a concentrated microvesicle fraction. In some embodiments, thenucleic acids are subsequently extracted from the concentratedmicrovesicle fraction.

In some embodiments, the capture surface is a membrane. In one aspect,the membrane comprises regenerated cellulose. For example, the membranehas a pore size at least 1 μm, such as for example, in a range between2-5 μm. In some embodiments, the membrane has a pore size in a rangebetween 3-5 μm. In some embodiments, the membrane comprisespolyethersulfone (PES).

In some embodiments, the membrane is charged. In some embodiments, themembrane is positively charged. In some embodiments, the membrane isnegatively charged.

In some aspects, the membrane is functionalized. For example, themembrane is functionalized with quaternary ammonium R—CH₂—N⁺(CH₃)₃.

In one embodiment, the capture surface comprises more than one membrane.In some embodiments, the capture surface comprises at least twomembranes, wherein each membrane is adjacently next to the othermembrane(s). In some embodiments, the capture surface comprises at leastthree membranes, wherein each of the three membranes is directlyadjacent to one another. In some embodiments, the capture surfacecomprises at least four membranes, wherein each of the four membranes isdirectly adjacent to one another.

In some embodiments, the capture surface is a bead. For example, thebead is magnetic. Alternatively, the bead is non-magnetic. In yetanother embodiment, the bead is functionalized with an affinity ligand.

In some embodiments, the capture surface is a slurry of polymer(s). Insome embodiments, the slurry of polymer(s) is shaped into a bead.

In some embodiments, the biological sample is plasma. In someembodiments, the biological sample is serum. In some embodiments, thebiological sample is urine. In some embodiments, the biological sampleis cerebrospinal fluid. In some embodiments, the biological sample iscell culture supernatant.

In some aspects, the method described herein further comprisescontacting the biological sample with a loading buffer. The loadingbuffer is in the range of pH 4-8. In one aspect, the loading buffer hasa neutral pH.

The methods described herein provide for the extraction of nucleic acidsfrom microvesicles. Preferably, the extracted nucleic acids are DNAand/or DNA and RNA. The extracted RNA may comprise messenger RNA,ribosomal RNA, transfer RNA, or small RNAs such as microRNAs, or anycombination thereof.

Various nucleic acid sequencing techniques are used to detect andanalyze nucleic acids such as cell free DNA and/or RNA extracted fromthe microvesicle fraction from biological samples. Analysis of nucleicacids such as cell free DNA and/or nucleic acids extracted frommicrovesicles for diagnostic purposes has wide-ranging implications dueto the non-invasive nature in which microvesicles can be easilycollected. Use of microvesicle analysis in place of invasive tissuebiopsies will positively impact patient welfare, improve the ability toconduct longitudinal disease monitoring, and improve the ability toobtain expression profiles even when tissue cells are not easilyaccessible (e.g., in ovarian or brain cancer patients).

In some embodiments, the present invention is directed to compositionsand methods for providing an in-process control for nucleic acidsequencing techniques, including, for example, next-generationsequencing (NGS) assays, to detect low-frequency sequence variants.These controls provide a number of technical advantages.

The biological sample is a bodily fluid. The bodily fluids can be fluidsisolated from anywhere in the body of the subject, preferably aperipheral location, including but not limited to, for example, blood,plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleuralfluid, nipple aspirates, lymph fluid, fluid of the respiratory,intestinal, and genitourinary tracts, tear fluid, saliva, breast milk,fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organsystem fluid, ascitic fluid, tumor cyst fluid, amniotic fluid andcombinations thereof. For example, the bodily fluid is urine, blood,serum, or cerebrospinal fluid.

In any of the foregoing methods, the nucleic acids are DNA and/or DNAand RNA. Examples of RNA include messenger RNAs, transfer RNAs,ribosomal RNAs, small RNAs (non-protein-coding RNAs, non-messengerRNAs), microRNAs, piRNAs, exRNAs, snRNAs and snoRNAs.

In any of the foregoing methods, the nucleic acids are isolated from orotherwise derived from a sample, including RNA isolated from themicrovesicle fraction of a sample.

In any of the foregoing methods, the nucleic acids are cell-free nucleicacids, also referred to herein as circulating nucleic acids. In someembodiments, the cell-free nucleic acids are DNA or RNA.

Various aspects and embodiments of the invention will now be describedin detail. It will be appreciated that modification of the details maybe made without departing from the scope of the invention. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representations as tothe contents of these documents are based on the information availableto the applicants and do not constitute any admission as to thecorrectness of the dates or contents of these documents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic demonstrating one embodiment of the RNA and DNAisolation protocol for isolating a microvesicle fraction, releasing themicrovesicle nucleic acids, and extracting RNA and DNA using twoseparate protocols.

FIG. 2 is a schematic demonstrating another embodiment of the RNA andDNA isolation protocol for isolating a microvesicle fraction, releasingthe microvesicle nucleic acids, and extracting RNA and DNA using asingle protocol.

FIG. 3 is a graph showing the effect of chloroform concentration inphase separation for isolating microvesicle RNA and DNA in a singleextraction, as demonstrated by detection of wild-type BRAF RNA and DNA.

FIG. 4 is a graph showing the effect of chloroform concentration inphase separation for isolating microvesicle RNA and DNA in a singleextraction, as demonstrated by detection of GAPDH RNA and DNA.

FIG. 5 is a graph showing that the adjustment of pH in phase separationinfluences the DNA extraction and detection.

FIG. 6 is a graph showing the effect of titration of sample volume ofcerebrospinal fluid (CSF) on microvesicle RNA extraction and detection.

FIG. 7 is a graph showing the comparison of detection of microvesicleRNA targets from ultracentrifugation and EXO60 isolation methods.

FIG. 8 is a graph showing the comparison of detection of microvesicleRNA targets from ultracentrifugation and EXO60 isolation methods fordifferent patient CSF samples. Patient samples are designated by patientID. Varying sample volumes were utilized. (*) indicates post-mortemsample.

FIG. 9 is a graph showing the effect of CSF sample volume (0.25 ml, 0.5ml, 1.0 ml and 2.0 ml) on different microvesicle RNA isolation andextraction methods. UC (ultracentrifugation), uCSC (urine filtrationmethod), and EXO60.

FIG. 10 is a series of bioanalyzer plots depicting the RNA profiles fromextraction from 2 different urine samples using the EXO70 protocolcompared to the urine circulating stem cell (uCSC) method.

FIG. 11 is a graph showing the correlation between RNA detection afterisolation and extraction by EXO70 compared to the urineCSC method.

FIG. 12 is two graphs showing the detection of different RNA targetsafter isolation and extraction by EXO70 or uCSC method. RNA wasextracted and analyzed from the isolated microvesicle fraction (EXO70 oruCSC) and the flow-through or supernatant fraction after isolation(EXO70 flow or uCSC flow). (A) mRNA targets; (B) miRNA targets.

FIGS. 13-223 are a series of graphs and illustrations depicting thesensitivity and specificity of the EXO52 DNA and RNA isolation andextraction methods, along with comparisons to commercially availablecirculating nucleic acid isolation kits, referred to herein ascommercially available CNA kits.

FIG. 13 is a schematic representation of studies designed to evaluateDNA extraction with and without PLG-tubes.

FIGS. 14, 15, 16, and 17 are a series of graphs depicting DNA extractionwith and without PLG-tubes using an initial method of DNA/RNA isolation(EXO52.1) and commercially available kits.

FIGS. 18 and 19 are a series of graphs depicting DNA extraction usingmethods of the disclosure versus a commercially available circulatingnucleic acid extraction kit.

FIG. 20 is a graph depicting the effect of chloroform titration on RNAand DNA isolation of phenol phase.

FIG. 21 is a schematic representation of studies designed to evaluatethe effect of chloroform titration on RNA isolation and DNA isolation ofphenol phase in PLG-tubes.

FIGS. 22, 23, 24, 25, and 26 are a series of graphs depicting theeffects of chloroform titration on RNA isolation (FIG. 22), RNA and DNAisolation (FIGS. 23, 24), and DNA isolation (FIGS. 25, 26).

FIG. 27 is a graph depicting DNA isolation using RNeasy protocol (w/oPLG tube) and chloroform titration.

FIG. 28 is a schematic representation of studies designed to evaluateDNA isolation without PLG-tubes and with a chloroform titration.

FIGS. 29, 30, and 31 are a series of graphs depicting DNA isolationusing RNeasy protocol (w/o PLG tube) and chloroform titration.

FIG. 32 is a graph depicting that adjusted chloroform additionco-isolates DNA and RNA.

FIG. 33 is a schematic representation of studies designed to evaluateDNA isolation without PLG-tubes and with a chloroform titration.

FIGS. 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44 are a series ofgraphs depicting DNA isolation using RNeasy protocol (w/o PLG tube) andchloroform titration.

FIG. 45 is a graph depicting the effect of pH changes in phaseseparation on DNA isolation.

FIG. 46 is a schematic representation of studies designed to evaluateDNA isolation from aqueous phase with a pH titration.

FIG. 47 is a schematic representation of the method of preparing pHconditioning solution.

FIG. 48 is a graph depicting Nala amplification curves for isolated RNAand DNA.

FIGS. 49, 50, 51, 52, 53, and 54 are a series of graphs depicting theeffect of pH titration on DNA isolation from aqueous phase.

FIG. 55 is a graph depicting that chloroform addition is the predominantfactor in determining the DNA content of the aqueous phase.

FIG. 56 is a graph depicting that RNA signal is not affected through theaddition of DNA isolation.

FIG. 57 is a schematic representation of studies designed to evaluateDNA isolation from aqueous phase with a chloroform titration and with orwithout adding pH solution.

FIG. 58 is a schematic representation of the method of preparing pHconditioning solution.

FIGS. 59, 60, 61, 62, 63, 64, 65, 66, 67, and 68 are a series of graphsdepicting the effect of chloroform titration with and without adding pHsolution on DNA isolation from aqueous phase.

FIG. 69 is a graph depicting the effect of a 4° C. or a room temperatureQiazol spin step on RNA isolation using a commercially available kit.

FIG. 70 is a graph depicting the effect of a 4° C. or a room temperatureQiazol spin step on the methods of the disclosure.

FIGS. 71 and 72 are a schematic representation and an overview ofstudies designed to evaluate RNA isolation using a commerciallyavailable kit with either a 4° C. or a room temperature Qiazol spinstep.

FIGS. 73, 74, and 75 are a series of graphs depicting the effect of a 4°C. or a room temperature Qiazol spin step on a commercially availablekit.

FIGS. 76 and 77 are a schematic representation and an overview ofstudies designed to evaluate RNA isolation using the EXO52 method witheither a 4° C. or a room temperature Qiazol spin step.

FIGS. 78 and 79 are a series of graphs depicting the effect of a 4° C.or a room temperature Qiazol spin step on the methods of the disclosure.

FIG. 80 is a schematic representation of studies designed to evaluatethe effect of varying ethanol volumes between 1.5× to 2.6×.

FIGS. 81 and 82 are a series of graphs depicting the effect of varyingethanol volumes between 1.5× to 2.6× on DNA and RNA isolation.

FIG. 83 is a graph depicting the results of ProtK digestion at roomtemperature before the binding step.

FIG. 84 is a schematic representation of studies designed to evaluateProtK digestion at room temperature before the binding step.

FIGS. 85 and 86 are a series of graphs depicting the results of ProtKdigestion at room temperature before the binding step.

FIG. 87 is a graph depicting that the loading capacity is over 8 mL ofplasma.

FIG. 88 is a graph depicting that the flow-through does not have abreakthrough point up to 8 mL of plasma.

FIG. 89 is a graph depicting different binding capacity for exosomes andnucleosomes.

FIG. 90 is a schematic representation of studies designed to evaluatethe loading capacity.

FIG. 91 is a graph depicting different binding capacity for exosomes andnucleosomes.

FIGS. 92 and 93 are a series of graphs depicting that the loadingcapacity is over 8 mL of plasma.

FIG. 94 is a graph depicting that the flow-through does not have abreakthrough point up to 8 mL of plasma.

FIGS. 95, 96, and 97 are a series of graphs depicting the effect ofvarying the loading volume of plasma on DNA and RNA isolation.

FIG. 98 is a graph depicting that the flow-through does not have abreakthrough point up to 8 mL of plasma.

FIGS. 99 and 100 are a series of graphs depicting the effect of varyingthe loading volume of plasma on DNA and RNA isolation.

FIGS. 101, 102, 103, 104, 105, and 106 are a series of graphs depictingdifferent binding capacity for exosomes and nucleosomes.

FIGS. 107 and 108 are a series of graphs depicting cell-free DNA (cfDNA)isolation using different isolation techniques including the methods ofthe disclosure and commercially available kits.

FIG. 109 is a schematic representation of studies designed to comparecfDNA isolating using methods of the disclosure with commerciallyavailable kits.

FIGS. 110 and 111 are a schematic representation and an overview ofstudies designed to compare cfDNA isolation using different isolationtechniques including methods of the disclosure and commerciallyavailable kits.

FIGS. 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 123 area series of graphs depicting cfDNA isolation using different isolationtechniques including the methods of the disclosure and commerciallyavailable kits.

FIGS. 124, 125, 126, 127, and 128 are a series of graphs and tablesdepicting a comparison of cfDNA copy number using different isolationtechniques including methods of the disclosure and commerciallyavailable kits.

FIGS. 129, 130, and 131 are a schematic representation and overviews ofstudies designed to evaluate use of the AllPrep Micro kit for downstreamanalysis of isolated DNA and RNA.

FIGS. 132, 133, 134, 135, and 136 are a series of graphs depicting theuse of the AllPrep Micro kit for downstream analysis of isolated DNA andRNA.

FIGS. 137 and 138 are a series of graphs depicting cell-free DNA (cfDNA)isolation using different isolation techniques including the methods ofthe disclosure and commercially available kits.

FIG. 139 is a schematic representation of studies designed to comparecfDNA isolated using methods of the disclosure and commerciallyavailable kits.

FIGS. 140, 141, 142, 143, 144, 145, and 146 are a series of graphsdepicting cell-free DNA (cfDNA) isolation using different isolationtechniques including the methods of the disclosure and commerciallyavailable kits.

FIGS. 147, 148, and 149 are schematic representations of studiesdesigned to compare cfDNA isolated using methods of the disclosure andcommercially available kits.

FIGS. 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, and 170 are a series of graphsdepicting cell-free DNA (cfDNA) isolation using different isolationtechniques including the methods of the disclosure and commerciallyavailable kits.

FIG. 171 is series of graphs depicting that the methods of thedisclosure consistently outperform the commercially available cNA kits.

FIGS. 172, 173, and 174 are schematic representations of studiesdesigned to compare cfDNA isolated using methods of the disclosure andcommercially available kits.

FIGS. 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, and 196 are a series of graphsdepicting cell-free DNA (cfDNA) isolation using different isolationtechniques including the methods of the disclosure and commerciallyavailable kits.

FIG. 197 is a graph depicting the effect of multiple separate Qiazolelution steps on DNA and RNA isolation.

FIG. 198 is a schematic representation of studies designed to evaluateDNA and RNA isolation using multiple Qiazol elution steps.

FIGS. 199, 200, 201, and 202 are a series of graphs depicting the effectof multiple separate Qiazol elution steps on DNA and RNA isolation.

FIG. 203 is a schematic representation of studies designed to evaluateDNA and RNA isolation using multiple Qiazol elution steps.

FIGS. 204, 205, and 206 are a series of graphs depicting the effect ofmultiple separate Qiazol elution steps on DNA and RNA isolation.

FIG. 207 is a graph depicting the effect of double RNeasy loading stepswith ethanol precipitation on DNA and RNA isolation.

FIGS. 208 and 209 are a schematic representation and an overview ofstudies designed to evaluate DNA and RNA isolation using double RNeasyloading steps with ethanol precipitation.

FIGS. 210 and 211 are a series of graphs depicting the effect of doubleRNeasy loading steps with ethanol precipitation on DNA and RNAisolation.

FIG. 212 is a graph depicting the effect of different downstream columnson DNA and RNA isolation.

FIG. 213 is a schematic representation of studies designed to evaluateDNA and RNA isolation using different downstream columns.

FIGS. 214, 215, 216, and 217 are a series of graphs depicting the effectof different downstream columns on DNA and RNA isolation.

FIG. 218 is a graph depicting the effect of multiple RNeasy elutionsteps on DNA and RNA isolation.

FIG. 219 is a schematic representation of studies designed to evaluateDNA and RNA isolation using multiple RNeasy elution steps.

FIGS. 220, 221, 222, and 223 are a series of graphs depicting the effectof multiple RNeasy elution steps on DNA and RNA isolation.

FIG. 224 is a series of graphs depicting the size distribution ofnucleic acids in plasma. Complete nucleic acid isolation from 1 mLplasma was subjected to either RNase A digestion (“cfDNA”), DNase Idigestion (“exoRNA”), or mock treatment (“EXO52”). After reactioncleanup, the size distribution of nucleic acids present in the isolationwas measured by a Bioanalyzer Pico 6000 assay.

FIG. 225 is a graph depicting sequential isolation of nucleic acids from2 ml of blood plasma. Blood plasma from a normal healthy donor waspassed through an EX052 column and the material left in the flow throughwas isolated using either a commercially available exoRNeasy kit (RNA)or a commercially available circulating nucleic acid kit (DNA). Theoverall yield is compared to EXO52 (RNA+DNA) using (RT)-qPCR againstBRAF, KRAS and 18S genes as a function of delta CT. Error bars representthree replicate isolations.

FIG. 226 is a series of graphs depicting exoRNA and ciDNA bothcontribute substantially to the total nucleic acids harvested from bloodplasma. 1 mL plasma from healthy donors was isolated using either thecommercially available exoRNeasy kit (RNA) or an EXO52 isolation with areverse transcription step (RNA+DNA) or without (DNA). Absolutequantification by RT-qPCR is presented as a boxplot and indicates themedian copy number per mL plasma with individual donors plotted asshapes.

FIGS. 227 and 228 are a series of graphs depicting the ability of theEXO52 methods provided herein to capture total circulating nucleicacids. The EXO52 methods were compared to a commercially availablecirculating nucleic acid DNA isolation kit.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of isolating cell-free DNA(cfDNA) and/or cfDNA and nucleic acids including at least RNA frommicrovesicles by capturing the DNA and the microvesicles to a surface,subsequently lysing the microvesicles to release the nucleic acids,particularly RNA, contained therein, and eluting the DNA and/or DNA andnucleic acids including at least RNA from the capture surface.Microvesicles are shed by eukaryotic cells, or budded off of the plasmamembrane, to the exterior of the cell. These membrane vesicles areheterogeneous in size with diameters ranging from about 10 nm to about5000 nm. All membrane vesicles shed by cells <0.8 μm in diameter arereferred to herein collectively as “microvesicles.” These microvesiclesinclude microvesicles, microvesicle-like particles, prostasomes,dexosomes, texosomes, ectosomes, oncosomes, apoptotic bodies,retrovirus-like particles, and human endogenous retrovirus (HERV)particles. Small microvesicles (approximately 10 to 1000 nm, and moreoften 30 to 200 nm in diameter) that are released by exocytosis ofintracellular multivesicular bodies are referred to in the art as“microvesicles.”

Current methods of isolating DNA and/or DNA and nucleic acids includingat least RNA from microvesicles include ultracentrifugation,ultrafiltration, e.g., using 100 kD filters, polymer precipitationtechniques, and/or filtration based on size. However, there exists aneed for alternative methods that are efficient and effective forisolating microvesicles and, optionally, extracting the nucleic acidscontained therein, preferably microvesicle RNA, for use in a variety ofapplications, including diagnostic purposes.

The isolation and extraction methods and/or kits provided hereinreferred to as the EXO52 DNA and/or DNA and RNA isolation methods and/orkits use a spin-column based purification process using an affinitymembrane that binds cell free DNA and/or microvesicles. The methods andkits of the disclosure allow for the capability to run large numbers ofclinical samples in parallel, using volumes from 0.2 up to 4 mL on asingle column. The cell-free DNA isolated using the EXO52 procedure ishighly pure. The isolated RNA is highly pure, protected by a vesiclemembrane until lysis, and intact vesicles can be eluted from the EXO52membrane. The EXO52 procedure is able to deplete substantially allcell-free DNA from plasma input, and is equal to or better in DNA yieldwhen compared to commercially available circulating DNA isolation kits.The EXO52 procedure is able to deplete substantially all mRNA fromplasma input, and is equal or better in mRNA/miRNA yield when comparedto ultracentrifugation or direct lysis. In contrast to commerciallyavailable kits and/or previous isolation methods, the EXO52 methodsand/or kits enrich for the microvesicle bound fraction of miRNAs, andthey are easily scalable to large amounts of input material. Thisability to scale up enables research on interesting, low abundanttranscripts. In comparison with other commercially available products onthe market, the methods and kits of the disclosure provide uniquecapabilities that are demonstrated by the examples provided herein.

The EXO52 methods and kits isolate and extract nucleic acids, e.g., DNAand/or DNA and nucleic acids including at least RNA from a biologicalsample using the following the general procedure. First, the sample,including the cfDNA and the microvesicle fraction, is bound to amembrane filter, and the filter is washed. Then, a phenol-based reagentis used to perform on-membrane lysis and release of the nucleic acids,e.g., DNA and/or DNA and RNA. Chloroform extraction is then performedusing PLG tubes, followed by ethanol conditioning. The nucleic acids,e.g., DNA and/or DNA and RNA, is then bound to a silica column, washedand then eluted. The extracted nucleic acids, e.g., DNA and/or DNA andRNA, can then be further analyzed, for example, using any of a varietyof downstream assays.

In some embodiments, the method includes the following steps. The filteris contained in spin column. Prior to addition of the lysis reagent, thesample is bound to a membrane filter in a spin column, and the spincolumn is then spun for 1 min at approximately 500×g. The flow-throughis then discarded, a buffer is added to the spin column, and the spincolumn is spun again for 5 min at approximately 5000×g to removeresidual volume from the column. The flow-through is discarded afterthis second spin. The spin column is then contacted with thephenol-based lysis reagent and spun for 5 min at approximately 5000×g tocollect the homogenate containing the lysed microvesicles and capturedcfDNA. In some embodiments, the lysis buffer is a phenol-based lysisbuffer. For example, the lysis buffer is QIAzol® lysis reagent (Qiagen).The homogenate is then subject to nucleic acid isolation and extraction.In some embodiments, a control for RNA isolation efficiency, such as,for example, Q-beta or any other control described herein, is spiked-into the homogenate prior to nucleic acid isolation and extraction.

In some embodiments, the nucleic acid is isolated according to thefollowing steps. After addition of the lysis reagent, chloroform is thenadded to the homogenate, and the solution is mixed vigorously for abrief time period. In some embodiments, 350 μl chloroform is added tothe homogenate. The solution is then centrifuged for 5 min at 12,000×gat 4° C. The upper aqueous phase is then transferred to a new collectiontube, and 2 volumes of 100% ethanol is added to the upper aqueous phase,and the solution is mixed. The solution can then be processed using anyof a variety of art-recognized methods for isolating and/or extractingnucleic acids.

The isolated nucleic acids, e.g., DNA and/or DNA and RNA, can then besubject to further analysis using any of a variety of downstream assays.In some embodiments, the combined detection of DNA and RNA is used toincrease the sensitivity for actionable mutations. There are multiplepotential sources of detectable mutations in circulating nucleic acids.For example, living tumor cells are a potential source for RNA and DNAisolated from the microvesicle fraction of a sample, and dying tumorcells are potential sources for cell-free DNA sources such as, forexample, apoptotic vesicle DNA and cell-free DNA from necrotic tumorcells. As mutated nucleic acids are relatively infrequent incirculation, the maximization of detection sensitivity becomes veryimportant. Combined isolation of DNA and RNA delivers comprehensiveclinical information to assess progression of disease and patientresponse to therapy. However, in contrast to the methods and kitsprovided herein, commercially available kits for detecting circulatingnucleic acids are only able to isolate cfDNA from plasma, i.e., fromdying cells. As shown in FIGS. 227-228, EXO52 captured all cfDNA, andEXO52 detected significantly more copies combining exoRNA and cfDNA vs.cfDNA alone. Those of ordinarily skill in the art will appreciate thatmore copies of a mutation or other biomarker leads to enhancedsensitivity and accuracy in identifying mutations and other biomarkers.

As used herein, the term “nucleic acids” refer to DNA and RNA. Thenucleic acids can be single stranded or double stranded. In someinstances, the nucleic acid is DNA. In some instances, the nucleic acidis RNA. RNA includes, but is not limited to, messenger RNA, transferRNA, ribosomal RNA, non-coding RNAs, microRNAs, and HERV elements.

As used herein, the term “biological sample” refers to a sample thatcontains biological materials such as DNA, RNA and protein.

In some embodiments, the biological sample may suitably comprise abodily fluid from a subject. The bodily fluids can be fluids isolatedfrom anywhere in the body of the subject, such as, for example, aperipheral location, including but not limited to, for example, blood,plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleuralfluid, nipple aspirates, lymph fluid, fluid of the respiratory,intestinal, and genitourinary tracts, tear fluid, saliva, breast milk,fluid from the lymphatic system, semen, intra-organ system fluid,ascitic fluid, tumor cyst fluid, amniotic fluid and cell culturesupernatant, and combinations thereof. Biological samples can alsoinclude fecal or cecal samples, or supernatants isolated therefrom.

In some embodiments, the biological sample may suitably comprise cellculture supernatant.

In some embodiments, the biological sample may suitably comprise atissue sample from a subject. The tissue sample can be isolated fromanywhere in the body of the subject.

A suitable sample volume of a bodily fluid is, for example, in the rangeof about 0.1 ml to about 30 ml fluid. The volume of fluid may depend ona few factors, e.g., the type of fluid used. For example, the volume ofserum samples may be about 0.1 ml to about 4 ml, preferably about 0.2 mlto 4 ml. The volume of plasma samples may be about 0.1 ml to about 4 ml,preferably 0.5 ml to 4 ml. The volume of urine samples may be about 10ml to about 30 ml, preferably about 20 ml.

While the examples provided herein used plasma samples, the skilledartisan will appreciate that these methods are applicable to a varietyof biological samples.

The methods and kits of the disclosure are suitable for use with samplesderived from a human subject. The methods and kits of the disclosure aresuitable for use with samples derived from a human subject. In addition,the methods and kits of the disclosure are also suitable for use withsamples derived from a human subject. The methods and kits of thedisclosure are suitable for use with samples derived from a non-humansubject such as, for example, a rodent, a non-human primate, a companionanimal (e.g., cat, dog, horse), and/or a farm animal (e.g., chicken).

The term “subject” is intended to include all animals shown to orexpected to have nucleic acid-containing particles. In particularembodiments, the subject is a mammal, a human or nonhuman primate, adog, a cat, a horse, a cow, other farm animals, or a rodent (e.g. mice,rats, guinea pig. etc.). A human subject may be a normal human beingwithout observable abnormalities, e.g., a disease. A human subject maybe a human being with observable abnormalities, e.g., a disease. Theobservable abnormalities may be observed by the human being himself, orby a medical professional. The term “subject,” “patient,” and“individual” are used interchangeably herein.

While the working examples provided herein use a membrane as the capturesurface, it should be understood that the format of the capturingsurface, e.g., beads or a filter (also referred to herein as amembrane), does not affect the ability of the methods provided herein toefficiently capture microvesicles from a biological sample.

While the examples provided herein use chloroform during the extractionstep, those of ordinary skill in the art will appreciate that anychemical that performs the same task as chloroform during nucleic acidextraction can be used in the methods provided herein. By way ofnon-limiting example, suitable chemicals for use in the extraction stepinclude dichloromethane, toluene, hexane, MTBE, and ethyl acetate(EtOAc).

A wide range of surfaces are capable of capturing microvesiclesaccording to the methods provided herein, but not all surfaces willcapture microvesicles (some surfaces do not capture anything).

The present disclosure also describes a device for isolating andconcentrating microvesicles from biological or clinical samples usingdisposable plastic parts and centrifuge equipment. For example, thedevice comprises a column comprising a capture surface (i.e., a membranefilter), a holder that secures the capture surface between the outerfrit and an inner tube, and a collection tube. The outer frit comprisesa large net structure to allow passing of liquid, and is preferably atone end of the column. The inner tube holds the capture surface inplace, and preferably is slightly conus-shaped. The collection tube maybe commercially available, i.e., 50 ml Falcon tube. The column ispreferably suitable for spinning, i.e., the size is compatible withstandard centrifuge and micro-centrifuge machines.

In embodiments where the capture surface is a membrane, the device forisolating the microvesicle fraction from a biological sample contains atleast one membrane. In some embodiments, the device comprises one, two,three, four, five or six membranes. In some embodiments, the devicecomprises three membranes. In embodiments where the device comprisesmore than one membrane, the membranes are all directly adjacent to oneanother at one end of the column. In embodiments where the devicecomprises more than one membrane, the membranes are all identical toeach other, i.e., are of the same charge and/or have the same functionalgroup.

It should be noted that capture by filtering through a pore size smallerthan the microvesicles is not the primary mechanism of capture by themethods provided herein. However, filter pore size is nevertheless veryimportant, e.g. because mRNA gets stuck on a 20 nm filter and cannot berecovered, whereas microRNAs can easily be eluted off, and e.g. becausethe filter pore size is an important parameter in available surfacecapture area.

The methods provided herein use any of a variety of capture surfaces. Insome embodiments, the capture surface is a membrane, also referred toherein as a filter or a membrane filter. In some embodiments, thecapture surface is a commercially available membrane. In someembodiments, the capture surface is a charged commercially availablemembrane. In some embodiments, the capture surface is neutral. In someembodiments, the capture surface is selected from Mustang® Ion ExchangeMembrane from PALL Corporation; Vivapure® Q membrane from Sartorius AG;Sartobind® Q, or Vivapure® Q Maxi H; Sartobind® D from Sartorius AG,Sartobind (S) from Sartorius AG, Sartobind® Q from Sartorius AG,Sartobind® IDA from Sartorius AG, Sartobind® Aldehyde from Sartorius AG,Whatman® DE81 from Sigma, Fast Trap Virus Purification column from EMDMillipore; Thermo Scientific* Pierce Strong Cation and Anion ExchangeSpin Columns.

In embodiments where the capture surface is charged, the capture surfacecan be a charged filter selected from the group consisting of 0.65 umpositively charged Q PES vacuum filtration (Millipore), 3-5 umpositively charged Q RC spin column filtration (Sartorius), 0.8 umpositively charged Q PES homemade spin column filtration (Pall), 0.8 umpositively charged Q PES syringe filtration (Pall), 0.8 um negativelycharged S PES homemade spin column filtration (Pall), 0.8 um negativelycharged S PES syringe filtration (Pall), and 50 nm negatively chargednylon syringe filtration (Sterlitech). Preferably, the charged filter isnot housed in a syringe filtration apparatus, as Qiazol/RNA is harder toget out of the filter in these embodiments. Preferably, the chargedfilter is housed at one end of a column.

In embodiments where the capture surface is a membrane, the membrane canbe made from a variety of suitable materials. In some embodiments, themembrane is polyethersulfone (PES) (e.g., from Millipore or PALL Corp.).In some embodiments, the membrane is regenerated cellulose (RC) (e.g.,from Sartorius or Pierce).

In some embodiments, the capture surface is a positively chargedmembrane. In some embodiments, the capture surface is a Q membrane,which is a positively charged membrane and is an anion exchanger withquaternary amines. For example, the Q membrane is functionalized withquaternary ammonium, R—CH₂—N⁺(CH₃)₃. In some embodiments, the capturesurface is a negatively charged membrane. In some embodiments, thecapture surface is an S membrane, which is a negatively charged membraneand is a cation exchanger with sulfonic acid groups. For example, the Smembrane is functionalized with sulfonic acid, R—CH₂SO₃ ⁻. In someembodiments, the capture surface is a D membrane, which is a weak basicanion exchanger with diethylamine groups, R—CH₂—NH⁺(C₂H₅)₂. In someembodiments, the capture surface is a metal chelate membrane. Forexample, the membrane is an IDA membrane, functionalized withminodiacetic acid —N(CH₂COOH⁻)₂. In some embodiments, the capturesurface is a microporous membrane, functionalized with aldehyde groups,—CHO. In other embodiments, the membrane is a weak basic anionexchanger, with diethylaminoethyl (DEAE) cellulose. Not all chargedmembranes are suitable for use in the methods provided herein, e.g., RNAisolated using Sartorius Vivapure S membrane spin column showed RT-qPCRinhibition and, thus, unsuitable for PCR related downstream assay.

In embodiments where the capture surface is charged, microvesicles canbe isolated with a positively charged filter.

In embodiments where the capture surface is charged, the pH duringmicrovesicle capture is a pH≤7. In some embodiments, the pH is greaterthan 4 and less than or equal to 8.

In embodiments where the capture surface is a positively charged Qfilter, the buffer system includes a wash buffer comprising 250 mM BisTris Propane, pH 6.5-7.0. In embodiments where the capture surface is apositively charged Q filter, the lysis buffer is Qiazol. In embodimentswhere the capture surface is a positively charged Q filter, the lysisbuffer is present at one volume. In embodiments where the capturesurface is a positively charged Q filter, the lysis buffer is present atmore than one volume.

Depending on the membrane material, the pore sizes of the membrane rangefrom 3 μm to 20 nm.

The surface charge of the capture surface can be positive, negative orneutral. In some embodiments, the capture surface is a positivelycharged bead or beads.

The methods provided herein include a lysis reagent. In someembodiments, the agent used for on-membrane lysis is a phenol-basedreagent. In some embodiments, the lysis reagent is a guanidinium-basedreagent. In some embodiments, the lysis reagent is a high salt basedbuffer. In some embodiments, the lysis reagent is QIAzol.

The methods provided herein include a variety of buffers includingloading and wash buffers. Loading and wash buffers can be of high or lowionic strength. The salt concentration, e.g., NaCl concentration, can befrom 0 to 2.4M. The buffers can include a variety of components. In someembodiments, the buffers include one or more of the followingcomponents: Tris, Bis-Tris, Bis-Tris-Propane, Imidazole, Citrate, MethylMalonic Acid, Acetic Acid, Ethanolamine, Diethanolamine, Triethanolamine(TEA) and Sodium phosphate. In the methods provided herein, the pH ofloading and wash buffers is important. Filters tend to clog when plasmasamples at set to pH≤5.5 before loading (the plasma will not spinthrough the column at all), and at higher pH microvesicle RNA recoveryis lower due to instability of the microvesicles. At neutral pH, the RNArecovery from microvesicles is optimal. In some embodiments, the bufferused is at 1× concentration, 2× concentration, 3× concentration, or 4×concentration. For example, the loading or binding buffer is at 2×concentration while the wash buffer is at 1× concentration.

In some embodiments, the methods include one or more wash steps, forexample, after contacting the biological sample with the capturesurface. In some embodiments, detergents are added to the wash buffer tofacilitate removing the non-specific binding (i.e., contaminants, celldebris, and circulating protein complexes or nucleic acids), to obtain amore pure microvesicle fraction. Detergents suitable for use include,but are not limited to, sodium dodecyl sulfate (SDS), Tween-20,Tween-80, Triton X-100, Nonidet P-40 (NP-40), Brij-35, Brij-58, octylglucoside, octyl thioglucoside, CHAPS or CHAPSO.

In some embodiments, the capture surface, e.g., membrane, is housedwithin a device used for centrifugation; e.g. spin columns, or forvacuum system e.g. vacuum filter holders, or for filtration withpressure e.g. syringe filters. In a preferred embodiment, the capturesurface is housed in a spin column or vacuum system.

The isolation of microvesicles from a biological sample prior toextraction of nucleic acids is advantageous for the followingreasons: 1) extracting nucleic acids from microvesicles provides theopportunity to selectively analyze disease or tumor-specific nucleicacids obtained by isolating disease or tumor-specific microvesiclesapart from other microvesicles within the fluid sample; 2) nucleicacid-containing microvesicles produce significantly higher yields ofnucleic acid species with higher integrity as compared to theyield/integrity obtained by extracting nucleic acids directly from thefluid sample without first isolating microvesicles; 3) scalability,e.g., to detect nucleic acids expressed at low levels, the sensitivitycan be increased by concentrating microvesicles from a larger volume ofsample using the methods described herein; 4) more pure or higherquality/integrity of extracted nucleic acids in that proteins, lipids,cell debris, cells and other potential contaminants and PCR inhibitorsthat are naturally found within biological samples are excluded beforethe nucleic acid extraction step; and 5) more choices in nucleic acidextraction methods can be utilized as isolated microvesicle fractionscan be of a smaller volume than that of the starting sample volume,making it possible to extract nucleic acids from these fractions orpellets using small volume column filters.

Several methods of isolating microvesicles from a biological sample havebeen described in the art. For example, a method of differentialcentrifugation is described in a paper by Raposo et al. (Raposo et al.,1996), a paper by Skog et. al. (Skog et al., 2008) and a paper byNilsson et. al. (Nilsson et al., 2009). Methods of ion exchange and/orgel permeation chromatography are described in U.S. Pat. Nos. 6,899,863and 6,812,023. Methods of sucrose density gradients or organelleelectrophoresis are described in U.S. Pat. No. 7,198,923. A method ofmagnetic activated cell sorting (MACS) is described in a paper by Taylorand Gercel Taylor (Taylor and Gercel-Taylor, 2008). A method ofnanomembrane ultrafiltration concentration is described in a paper byCheruvanky et al. (Cheruvanky et al., 2007). A method of Percollgradient isolation is described in a publication by Miranda et al.(Miranda et al., 2010). Further, microvesicles may be identified andisolated from bodily fluid of a subject by a microfluidic device (Chenet al., 2010). In research and development, as well as commercialapplications of nucleic acid biomarkers, it is desirable to extract highquality nucleic acids from biological samples in a consistent, reliable,and practical manner.

An object of the present invention is therefore to provide a method forquick and easy isolation of nucleic acid-containing particles frombiological samples such as body fluids and extraction of high qualitynucleic acids from the isolated particles. The method of the inventionmay be suitable for adaptation and incorporation into a compact deviceor instrument for use in a laboratory or clinical setting, or in thefield.

In some embodiments, the sample is not pre-processed prior to isolationand extraction of nucleic acids, e.g., DNA and/or DNA and RNA, from thebiological sample.

In some embodiments, the sample is subjected to a pre-processing stepprior to isolation, purification or enrichment of the microvesicles isperformed to remove large unwanted particles, cells and/or cell debrisand other contaminants present in the biological sample. Thepre-processing steps may be achieved through one or more centrifugationsteps (e.g., differential centrifugation) or one or more filtrationsteps (e.g., ultrafiltration), or a combination thereof. Where more thanone centrifugation pre-processing steps are performed, the biologicalsample may be centrifuged first at the lower speed and then at thehigher speed. If desired, further suitable centrifugation pre-processingsteps may be carried out. Alternatively or in addition to the one ormore centrifugation pre-processing steps, the biological sample may befiltered. For example, a biological sample may be first centrifuged at20,000 g for 1 hour to remove large unwanted particles; the sample canthen be filtered, for example, through a 0.8 μm filter.

In some embodiments, the sample is pre-filtered to exclude particleslarger than 0.8 μm. In some embodiments, the sample includes an additivesuch as EDTA, sodium citrate, and/or citrate-phosphate-dextrose.Preferably, the sample does not contain heparin, as heparin cannegatively impact RT-qPCR and other nucleic acid analysis. In someembodiments, the sample is mixed with a buffer prior to purificationand/or nucleic acid isolation and/or extraction. In some embodiments,the buffer is XBP buffer.

In some embodiments, one or more centrifugation steps are performedbefore or after contacting the biological sample with the capturesurface to separate microvesicles and concentrate the microvesiclesisolated from the biological fraction. For example, the sample iscentrifuged at 20,000 g for 1 hour at 4° C. To remove large unwantedparticles, cells, and/or cell debris, the samples may be centrifuged ata low speed of about 100-500 g, preferably about 250-300 g.Alternatively or in addition, the samples may be centrifuged at a higherspeed. Suitable centrifugation speeds are up to about 200,000 g; forexample from about 2,000 g to less than about 200,000 g. Speeds of aboveabout 15,000 g and less than about 200,000 g or above about 15,000 g andless than about 100,000 g or above about 15,000 g and less than about50,000 g are preferred. Speeds of from about 18,000 g to about 40,000 gor about 30,000 g; and from about 18,000 g to about 25,000 g are morepreferred. Particularly preferred is a centrifugation speed of about20,000 g. Generally, suitable times for centrifugation are from about 5minutes to about 2 hours, for example, from about 10 minutes to about1.5 hours, or more preferably from about 15 minutes to about 1 hour. Atime of about 0.5 hours may be preferred. It is sometimes preferred tosubject the biological sample to centrifugation at about 20,000 g forabout 0.5 hours. However the above speeds and times can suitably be usedin any combination (e.g., from about 18,000 g to about 25,000 g, or fromabout 30,000 g to about 40,000 g for about 10 minutes to about 1.5hours, or for about 15 minutes to about 1 hour, or for about 0.5 hours,and so on). The centrifugation step or steps may be carried out atbelow-ambient temperatures, for example at about 0-10° C., preferablyabout 1-5° C., e.g., about 3° C. or about 4° C.

In some embodiments, one or more filtration steps are performed beforeor after contacting the biological sample with the capture surface. Afilter having a size in the range about 0.1 to about 1.0 μm may beemployed, preferably about 0.8 μm or 0.22 μm. The filtration may also beperformed with successive filtrations using filters with decreasingporosity.

In some embodiments, one or more concentration steps are performed, inorder to reduce the volumes of sample to be treated during thechromatography stages, before or after contacting the biological samplewith the capture surface. Concentration may be through centrifugation ofthe sample at high speeds, e.g. between 10,000 and 100,000 g, to causethe sedimentation of the microvesicles. This may consist of a series ofdifferential centrifugations. The microvesicles in the pellet obtainedmay be reconstituted with a smaller volume and in a suitable buffer forthe subsequent steps of the process. The concentration step may also beperformed by ultrafiltration. In fact, this ultrafiltration bothconcentrates the biological sample and performs an additionalpurification of the microvesicle fraction. In another embodiment, thefiltration is an ultrafiltration, preferably a tangentialultrafiltration. Tangential ultrafiltration consists of concentratingand fractionating a solution between two compartments (filtrate andretentate), separated by membranes of determined cut-off thresholds. Theseparation is carried out by applying a flow in the retentatecompartment and a transmembrane pressure between this compartment andthe filtrate compartment. Different systems may be used to perform theultrafiltration, such as spiral membranes (Millipore, Amicon), flatmembranes or hollow fibers (Amicon, Millipore, Sartorius, Pall, GF,Sepracor). Within the scope of the invention, the use of membranes witha cut-off threshold below 1000 kDa, preferably between 100 kDa and 1000kDa, or even more preferably between 100 kDa and 600 kDa, isadvantageous.

In some embodiments, one or more size-exclusion chromatography step orgel permeation chromatography steps are performed before or aftercontacting the biological sample with the capture surface. To performthe gel permeation chromatography step, a support selected from silica,acrylamide, agarose, dextran, ethylene glycol-methacrylate co-polymer ormixtures thereof, e.g., agarose-dextran mixtures, are preferably used.For example, such supports include, but are not limited to: SUPERDEX®200HR (Pharmacia), TSK G6000 (TosoHaas) or SEPHACRYL® S (Pharmacia).

In some embodiments, one or more affinity chromatography steps areperformed before or after contacting the biological sample with thecapture surface. Some microvesicles can also be characterized by certainsurface molecules. Because microvesicles form from budding of the cellplasma membrane, these microvesicles often share many of the samesurface molecules found on the cells they originated from. As usedherein, “surface molecules” refers collectively to antigens, proteins,lipids, carbohydrates, and markers found on the surface or in or on themembrane of the microvesicle. These surface molecules can include, forexample, receptors, tumor-associated antigens, membrane proteinmodifications (e.g., glycosylated structures). For example,microvesicles that bud from tumor cells often display tumor-associatedantigens on their cell surface. As such, affinity chromatography oraffinity exclusion chromatography can also be utilized in combinationwith the methods provided herein to isolate, identify, and or enrich forspecific populations of microvesicles from a specific donor cell type(Al-Nedawi et al., 2008; Taylor and Gercel-Taylor, 2008). For example,tumor (malignant or non-malignant) microvesicles carry tumor-associatedsurface antigens and may be detected, isolated and/or enriched via thesespecific tumor-associated surface antigens. In one example, the surfaceantigen is epithelial cell adhesion molecule (EpCAM), which is specificto microvesicles from carcinomas of long, colorectal, breast, prostate,head and neck, and hepatic origin, but not of hematological cell origin(Balzar et al., 1999; Went et al., 2004). Additionally, tumor-specificmicrovesicles can also be characterized by the lack of certain surfacemarkers, such as CD80 and CD86. In these cases, microvesicles with thesemarkers may be excluded for further analysis of tumor specific markers,e.g., by affinity exclusion chromatography. Affinity chromatography canbe accomplished, for example, by using different supports, resins,beads, antibodies, aptamers, aptamer analogs, molecularly imprintedpolymers, or other molecules known in the art that specifically targetdesired surface molecules on microvesicles.

Optionally, control particles may be added to the sample prior tomicrovesicle isolation or nucleic acid extraction to serve as aninternal control to evaluate the efficiency or quality of microvesiclepurification and/or nucleic acid extraction. The methods describedherein provide for the efficient isolation and the control particlesalong with the microvesicle fraction. These control particles includeQ-beta bacteriophage, virus particles, or any other particle thatcontains control nucleic acids (e.g., at least one control target gene)that may be naturally occurring or engineered by recombinant DNAtechniques. In some embodiments, the quantity of control particles isknown before the addition to the sample. The control target gene can bequantified using real-time PCR analysis. Quantification of a controltarget gene can be used to determine the efficiency or quality of themicrovesicle purification or nucleic acid extraction processes.

Preferably, the control particle is a Q-beta bacteriophage, referred toherein as “Q-beta particle.” The Q-beta particle used in the methodsdescribed herein may be a naturally-occurring virus particle or may be arecombinant or engineered virus, in which at least one component of thevirus particle (e.g., a portion of the genome or coat protein) issynthesized by recombinant DNA or molecular biology techniques known inthe art. Q-beta is a member of the leviviridae family, characterized bya linear, single-stranded RNA genome that consists of 3 genes encodingfour viral proteins: a coat protein, a maturation protein, a lysisprotein, and RNA replicase. Due to its similar size to averagemicrovesicles, Q-beta can be easily purified from a biological sampleusing the same purification methods used to isolate microvesicles, asdescribed herein. In addition, the low complexity of the Q-beta viralsingle-stranded gene structure is advantageous for its use as a controlin amplification-based nucleic acid assays. The Q-beta particle containsa control target gene or control target sequence to be detected ormeasured for the quantification of the amount of Q-beta particle in asample. For example, the control target gene is the Q-beta coat proteingene. After addition of the Q-beta particles to the biological sample,the nucleic acids from the Q-beta particle are extracted along with thenucleic acids from the biological sample using the extraction methodsdescribed herein. Detection of the Q-beta control target gene can bedetermined by RT-PCR analysis, for example, simultaneously with thebiomarker(s) of interest. A standard curve of at least 2, 3, or 4 knownconcentrations in 10-fold dilution of a control target gene can be usedto determine copy number. The copy number detected and the quantity ofQ-beta particle added can be compared to determine the quality of theisolation and/or extraction process.

In a preferred embodiment, the Q-beta particles are added to the urinesample prior to nucleic extraction. For example, the Q-beta particlesare added to the urine sample prior to ultrafiltration and/or after thepre-filtration step.

In some embodiments, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,1,000 or 5,000 copies of Q-beta particles added to a bodily fluidsample. In a preferred embodiment, 100 copies of Q-beta particles areadded to a bodily fluid sample. The copy number of Q-beta particles canbe calculated based on the ability of the Q-beta bacteriophage to infecttarget cells. Thus, the copy number of Q-beta particles is correlated tothe colony forming units of the Q-beta bacteriophage.

Nucleic Acid Extraction

The present invention is directed towards the use of a capture surfacefor the improved isolation, purification, or enrichment ofmicrovesicles. The methods disclosed herein provide a highly enrichedmicrovesicle fraction for extraction of high quality nucleic acids fromsaid microvesicles. The nucleic acid extractions obtained by the methodsdescribed herein may be useful for various applications in which highquality nucleic acid extractions are required or preferred, such as foruse in the diagnosis, prognosis, or monitoring of diseases or medicalconditions.

Recent studies reveal that nucleic acids within microvesicles have arole as biomarkers. For example, WO 2009/100029 describes, among otherthings, the use of nucleic acids extracted from microvesicles in GBMpatient serum for medical diagnosis, prognosis and therapy evaluation.WO 2009/100029 also describes the use of nucleic acids extracted frommicrovesicles in human urine for the same purposes. The use of nucleicacids extracted from microvesicles is considered to potentiallycircumvent the need for biopsies, highlighting the enormous diagnosticpotential of microvesicle biology (Skog et al., 2008).

The quality or purity of the isolated microvesicles can directly affectthe quality of the extracted microvesicle nucleic acids, which thendirectly affects the efficiency and sensitivity of biomarker assays fordisease diagnosis, prognosis, and/or monitoring. Given the importance ofaccurate and sensitive diagnostic tests in the clinical field, methodsfor isolating highly enriched microvesicle fractions from biologicalsamples are needed. To address this need, the present invention providesmethods for isolating microvesicles from biological sample for theextraction of high quality nucleic acids from a biological sample. Asshown herein, highly enriched microvesicle fractions are isolated frombiological samples by methods described herein, and wherein high qualitynucleic acids subsequently extracted from the highly enrichedmicrovesicle fractions. These high quality extracted nucleic acids areuseful for measuring or assessing the presence or absence of biomarkersfor aiding in the diagnosis, prognosis, and/or monitoring of diseases orother medical conditions.

As used herein, the term “high quality” in reference to nucleic acidextraction means an extraction in which one is able to detect 18S and28S rRNA, preferably in a ratio of approximately 1:1 to approximately1:2; and more preferably, approximately 1:2. Ideally, high qualitynucleic acid extractions obtained by the methods described herein willalso have an RNA integrity number of greater than or equal to 5 for alow protein biological sample (e.g., urine), or greater than or equal to3 for a high protein biological sample (e.g., serum), and a nucleic acidyield of greater than or equal to 50 pg/ml from a 20 ml low proteinbiological sample or a 1 ml high protein biological sample.

High quality RNA extractions are desirable because RNA degradation canadversely affect downstream assessment of the extracted RNA, such as ingene expression and mRNA analysis, as well as in analysis of non-codingRNA such as small RNA and microRNA. The new methods described hereinenable one to extract high quality nucleic acids from microvesiclesisolated from a biological sample so that an accurate analysis ofnucleic acids within the microvesicles can be performed.

Following the isolation of microvesicles from a biological sample,nucleic acid may be extracted from the isolated or enriched microvesiclefraction. To achieve this, in some embodiments, the microvesicles mayfirst be lysed. The lysis of microvesicles and extraction of nucleicacids may be achieved with various methods known in the art. In someembodiments, the nucleic acid extraction may be achieved usingphenol:chloroform according to standard procedures and techniques knownin the art. Such methods may also utilize a nucleic acid-binding columnto capture the nucleic acids contained within the microvesicles. Oncebound, the nucleic acids can then be eluted using a buffer or solutionsuitable to disrupt the interaction between the nucleic acids and thebinding column, thereby successfully eluting the nucleic acids.

In some embodiments, the nucleic acid extraction methods also includethe step of removing or mitigating adverse factors that prevent highquality nucleic acid extraction from a biological sample. Such adversefactors are heterogeneous in that different biological samples maycontain various species of adverse factors. In some biological samples,factors such as excessive DNA may affect the quality of nucleic acidextractions from such samples. In other samples, factors such asexcessive endogenous RNase may affect the quality of nucleic acidextractions from such samples. Many agents and methods may be used toremove these adverse factors. These methods and agents are referred tocollectively herein as an “extraction enhancement operations.” In someinstances, the extraction enhancement operation may involve the additionof nucleic acid extraction enhancement agents to the biological sample.To remove adverse factors such as endogenous RNases, such extractionenhancement agents as defined herein may include, but are not limitedto, an RNase inhibitor such as Superase-In (commercially available fromAmbion Inc.) or RNaseINplus (commercially available from Promega Corp.),or other agents that function in a similar fashion; a protease (whichmay function as an RNase inhibitor); DNase; a reducing agent; a decoysubstrate such as a synthetic RNA and/or carrier RNA; a soluble receptorthat can bind RNase; a small interfering RNA (siRNA); an RNA bindingmolecule, such as an anti-RNA antibody, a basic protein or a chaperoneprotein; an RNase denaturing substance, such as a high osmolaritysolution, a detergent, or a combination thereof.

For example, the extraction enhancement operation may include theaddition of an RNase inhibitor to the biological sample, and/or to theisolated microvesicle fraction, prior to extracting nucleic acid;preferably the RNase inhibitor has a concentration of greater than 0.027AU (I ×) for a sample equal to or more than 1 μl in volume;alternatively, greater than or equal to 0. 135 AU (5×) for a sampleequal to or more than 1 μl; alternatively, greater than or equal to 0.27AU (10×) for a sample equal to or more than I μl; alternatively, greaterthan or equal to 0.675 AU (25×) for a sample equal to or more than 1 μl;and alternatively, greater than or equal to 1.35 AU (50×) for a sampleequal to or more than 1 μl; wherein the I × concentration refers to anenzymatic condition wherein 0.027 AU or more RNase inhibitor is used totreat microvesicles isolated from 1 μl or more bodily fluid, the 5×concentration refers to an enzymatic condition wherein 0.135 AU or moreRNase inhibitor is used to treat microvesicles isolated from 1 μl ormore bodily fluid, the 10× protease concentration refers lo an enzymaticcondition wherein 0.27 AU or more RNase inhibitor is used to treatparticles isolated from 1 μl or more bodily fluid, the 25× concentrationrefers to an enzymatic condition wherein 0.675 AU or more RNaseinhibitor is used to treat microvesicles isolated from 1 μl or morebodily fluid, and the 50× protease concentration refers to an enzymaticcondition wherein 1.35 AU or more RNase inhibitor is used to treatparticles isolated from 1 μl or more bodily fluid. Preferably, the RNaseinhibitor is a protease, in which case, 1 AU is the protease activitythat releases folin-positive amino acids and peptides corresponding to 1μmol tyrosine per minute.

These enhancement agents may exert their functions in various ways,e.g., through inhibiting RNase activity (e.g., RNase inhibitors),through a ubiquitous degradation of proteins (e.g., proteases), orthrough a chaperone protein (e.g., a RNA-binding protein) that binds andprotects RNAs. In all instances, such extraction enhancement agentsremove or at least mitigate some or all of the adverse factors in thebiological sample or associated with the isolated particles that wouldotherwise prevent or interfere with the high quality extraction ofnucleic acids from the isolated particles.

In some embodiments, the quantification of 18S and 28S rRNAs extractedcan be used determine the quality of the nucleic acid extraction.

Detection of Nucleic Acid Biomarkers

In some embodiments, the extracted nucleic acid comprises DNA and/or DNAand RNA. In embodiments where the extracted nucleic acid comprises DNAand RNA, the RNA is preferably reverse-transcribed into complementaryDNA (cDNA) before further amplification. Such reverse transcription maybe performed alone or in combination with an amplification step. Oneexample of a method combining reverse transcription and amplificationsteps is reverse transcription polymerase chain reaction (RT-PCR), whichmay be further modified to be quantitative, e.g., quantitative RT-PCR asdescribed in U.S. Pat. No. 5,639,606, which is incorporated herein byreference for this teaching. Another example of the method comprises twoseparate steps: a first of reverse transcription to convert RNA intocDNA and a second step of quantifying the amount of cDNA usingquantitative PCR. As demonstrated in the examples that follow, the RNAsextracted from nucleic acid-containing particles using the methodsdisclosed herein include many species of transcripts including, but notlimited to, ribosomal 18S and 28S rRNA, microRNAs, transfer RNAs,transcripts that are associated with diseases or medical conditions, andbiomarkers that are important for diagnosis, prognosis and monitoring ofmedical conditions.

For example, RT-PCR analysis determines a Ct (cycle threshold) value foreach reaction. In RT-PCR, a positive reaction is detected byaccumulation of a fluorescence signal. The Ct value is defined as thenumber of cycles required for the fluorescent signal to cross thethreshold (i.e., exceeds background level). Ct levels are inverselyproportional to the amount of target nucleic acid, or control nucleicacid, in the sample (i.e., the lower the Ct level, the greater theamount of control nucleic acid in the sample).

In another embodiment, the copy number of the control nucleic acid canbe measured using any of a variety of art-recognized techniques,including, but not limited to, RT-PCR. Copy number of the controlnucleic acid can be determined using methods known in the art, such asby generating and utilizing a calibration, or standard curve.

In some embodiments, one or more biomarkers can be one or a collectionof genetic aberrations, which is used herein to refer to the nucleicacid amounts as well as nucleic acid variants within the nucleicacid-containing particles. Specifically, genetic aberrations include,without limitation, over-expression of a gene (e.g., an oncogene) or apanel of genes, under-expression of a gene (e.g., a tumor suppressorgene such as p53 or RB) or a panel of genes, alternative production ofsplice variants of a gene or a panel of genes, gene copy number variants(CNV) (e.g., DNA double minutes) (Hahn, 1993), nucleic acidmodifications (e.g., methylation, acetylation and phosphorylations),single nucleotide polymorphisms (SNPs), chromosomal rearrangements(e.g., inversions, deletions and duplications), and mutations(insertions, deletions, duplications, missense, nonsense, synonymous orany other nucleotide changes) of a gene or a panel of genes, whichmutations, in many cases, ultimately affect the activity and function ofthe gene products, lead to alternative transcriptional splice variantsand/or changes of gene expression level, or combinations of any of theforegoing.

The analysis of nucleic acids present in the isolated particles isquantitative and/or qualitative. For quantitative analysis, the amounts(expression levels), either relative or absolute, of specific nucleicacids of interest within the isolated particles are measured withmethods known in the art (described below). For qualitative analysis,the species of specific nucleic acids of interest within the isolatedmicrovesicles, whether wild type or variants, are identified withmethods known in the art.

The present invention also includes various uses of the new methods ofisolating microvesicles from a biological sample for high qualitynucleic acid extraction from a for (i) aiding in the diagnosis of asubject, (ii) monitoring the progress or reoccurrence of a disease orother medical condition in a subject, or (iii) aiding in the evaluationof treatment efficacy for a subject undergoing or contemplatingtreatment for a disease or other medical condition; wherein the presenceor absence of one or more biomarkers in the nucleic acid extractionobtained from the method is determined, and the one or more biomarkersare associated with the diagnosis, progress or reoccurrence, ortreatment efficacy, respectively, of a disease or other medicalcondition.

Kits for Isolating Microvesicles from a Biological Sample

One aspect of the present invention is further directed to kits for usein the methods disclosed herein. The kit comprises a capture surfaceapparatus sufficient to separate microvesicles from a biological samplefrom unwanted particles, debris, and small molecules that are alsopresent in the biological sample. The present invention also optionallyincludes instructions for using the foregoing reagents in the isolationand optional subsequent nucleic acid extraction process.

EXAMPLES

While the examples provided herein use a variety of membranes anddevices used for centrifugation and/or filtration purposes, it is to beunderstood that these methods can be used with any capture surfaceand/or housing device that allows for the efficient capture ofmicrovesicles and release of the nucleic acids, particularly, RNA,contained therein.

Example 1: EXO52 Isolation of DNA, as Well as Co-Isolation of RNA andDNA

This example demonstrates the ability of the EXO52 method to isolate allDNA from a plasma sample. It should be noted that in some of the Figurespresented herein, various terminology has been used to identifyprecursor methods to the isolation methods referred to herein as EXO52.For example, some Figures include terms such as old EXO52, EXO52.1, andvariations thereof. These earlier versions are provided solely as acomparison and to demonstrate the superior isolation achieved using theEXO52 methods of the disclosure. The use of the term EXO52.2 is theEXO52 method where the RNA and DNA extraction is performed in a singletube.

The EXO52 column can also be used to isolate all DNA from a plasmasample. Two methods for utilizing the EXO52 column for DNA isolation inaddition to RNA are depicted in FIG. 1 and FIG. 2. Specifically, thedifference between the two processes is that the RNA and DNA extractionis combined in one tube in EXO52, for ease in usability, streamlining ofthe protocol, and increased reproducibility. FIG. 3 shows a gain of 1.5Cts in EXO50 RNA+DNA (EXO52). EXO50 is a method for isolating RNA frommicrovesicles in a biological sample such as, for example, plasma. Thismethod is described in PCT Publication No. WO2014/107571. FIG. 4 showsthat increasing the amount of chloroform during phase separation addsthe DNA back to the aqueous phase, such that the DNA is co-isolated withthe normal EXO50 procedure. Further optimization of pH levels duringphase separation also adds the DNA to the prep, as shown in FIG. 5.

Thus, the methods of the disclosure can be used to isolate all DNA fromplasma samples. The DNA is recovered from the lower, hydrophobic phaseof the QIAzol lysis after phase separation. The methods of thedisclosure (e.g., two tubes or a single tube as in EXO52), separate RNAand DNA at similar levels for the same sample volume, and the RNA andDNA can be separated from each other. These methods of the disclosurecapture the same or more mRNA and much more miRNA than a commerciallyavailable isolation kit, e.g., Qiagen.

EXO52 can also be used for co-purification of RNA and DNA. As usedherein, EXO52 refers to the following protocol, unless otherwisespecified.

Sample Preparation: The EXO52 procedure can be used to isolate RNA andDNA from exosomes and other microvesicles using 0.2-4 mL of plasma orserum. It is recommended to only use pre-filtered plasma or serum,excluding particles larger than 0.8 μm. The list of compatible plasmatubes includes plasma with the additives EDTA, sodium citrate, andcitrate-phosphate-dextrose. Plasma containing heparin can inhibitRT-qPCR.

The sample, alone or diluted with a binding buffer, is then loaded ontothe EXO52 spin column and spun for 1 min at 500×g. Discard theflow-through and place the column back into the same collection tube.Wash buffer is then added and the EXO52 column is spun for 5 min at5000×g to remove residual volume from the column. Note: Aftercentrifugation, the EXO52 spin column is removed from the collectiontube so that the column does not contact the flow-through. The spincolumn is then transferred to a fresh collection tube, and 700 μL Qiazolis added to the membrane. Then, the spin column is spun for 5 min at5000×g to collect the homogenate containing the lysed exosomes. Thehomogenate is then transferred to a PLG tube.

Then, 350 μl chloroform is added to the tube containing the homogenateand shaken vigorously for 15 s. The tube containing the homogenate isthen kept at room temperature for 2-3 min, followed by centrifugationfor 5 min at 12,000×g at 4° C. After centrifugation, the centrifuge isheated up to room temperature (15-25° C.) if the same centrifuge will beused for the next centrifugation steps.

The upper aqueous phase is transferred to a new collection tube,avoiding transfer of any interphase material. 2 volumes of 100% ethanolare then added and mixed thoroughly by pipetting up and down severaltimes and without the use of a centrifuge. 700 μl of the sample,including any precipitate that may have formed, is then pipeted up tointo an RNeasy MinElute spin column in a 2 ml collection tube (Cat.#1026497), followed by centrifugation at ≥8000×g (≥10,000 rpm) for 15 sat room temperature (15-25° C.). The flow-through is discarded. Thesesteps are repeated with the remaining of the sample, and theflow-through is discarded.

EXO52 is useful for isolating and detecting DNA from biological samples.Vesicle RNA is thought to be derived from living cells in e.g. thediseased tissue. Cell-free DNA cfDNA) is thought to be derived fromdying cells e.g. necrotic cells in the disease tissue. Thus, cfDNA isuseful as an indicator of therapeutic response, while the RNA is anindicator of resistance mutations on the rise.

EXO52 is useful for detection of rare mutations in blood, as EXO52provides a sufficiently sensitive method that can be applied on nucleicacids of sufficient amount. The amount of actual DNA and RNA moleculesin biofluids is very limited, and EXO52 provides an isolation methodthat extracts all molecules of the blood that are relevant for mutationdetection in a volume small enough for effective downstream processingand/or analysis.

FIGS. 13-223 (referred to in only this example as “the Figures”)demonstrate the specificity and sensitivity of the EXO52 methods.

Studies have shown that the EXO50/52 column binds all DNA in plasma, butthe procedure to get the DNA out of the phenol phase does not producesatisfactory results, as the isolation procedure is very variable. Themethods provided herein allow for reproducible and efficient isolationand/or extraction of DNA from the membrane of the EXO50/52 column.

Two out of three replicates of Exo52 isolation with PLG tube show almostthe same CT values as a commercially available cfDNA KIT. One out ofthree replicates of EXO52 isolation without PLG tube show almost same CTvalues as cfDNA KIT. Depleted Plasma (Exo50 flow through of plasmawithout 2× Binding buffer) do not contain a lot of DNA (18S˜9CTdifference to normal isolation). Almost all almost all DNA bound to theExo50 column.

As shown in the Figures, adding chloroform to the EXO52 method allowedfor the co-isolation of both RNA and DNA, and adding chloroform did notharm the detection or isolation of RNA.

With regard to RNA isolation, it was determined that adding morechloroform did not influence RNA isolation if using RNA specific assays.DNA specific assays will result in lower CTs when adding more chloroformbecause DNA is in aqueous phase.

With regard to DNA isolation, CTs for DNA detecting assays increased inaqueous phase isolation with adding more chloroform and decreased inEXO52 phenol phase DNA isolation.

EXO50 isolation from phenol phase resulted in lowest CT values (=highestDNA yield). 90 μl chloroform resulted in best DNA yield from phenolphase.

Also without the PLG tube, the chloroform ratio at which no DNAcontamination was observed was approximately 0.13×.

As shown in the Figures, 350 μL of chloroform was sufficient to add allDNA from the EXO52 column to the aqueous phase during PC extraction.Higher chloroform amounts may interfere with RNA isolation.

As shown in the Figures, DNA yield increased in aqueous phase withadding more chloroform, whereas DNA yield decreased in phenol phase(EXO52 DNA Isolation). DNA isolation from aqueous phase yield more DNAcompared to EXO52 DNA Isolation from phenol phase. Little DNA appearedto stay in phenol phase or in remaining aqueous phase after removingupper phase, as it is difficult to remove whole phase w/o using PLGtube. DNA yield is similar in RT reaction (10 μl EXO50 eluate in final20 μl RT Mix) and 1:2 diluted EXO50 eluate. DNA did not seem to reactwith reverse transcription mix.

Studies were repeating using an RNA only GAPDH assay to see if the RNAonly GAPDH assay was affected by increasing chloroform addition. RNA wasnot affected with increasing chloroform addition. Studies were also runusing a GAPDH_RNA_DNA assay, which showed no replacement of RNA signalby the DNA (˜2CTs difference).

The BRAF assay showed a 2× increase in signal in the EXO50 RNA fractionby having DNA present in the aqueous phase. The GAPDH assay did not showa clear additive effect of DNA in the EXO50 RNA fraction since the addedcopies were minute in comparison to the RNA copies. With this cleardifference between RNA and DNA copies, no replacement of RNA signal canbe shown.

Studies were run to determine the effect, if any, of pH changes in phaseseparation. Adjustment of pH provides an alternative tool for adding DNAto the aqueous phase. It was found that too high of a pH interfered withRNA isolation.

High pH seemed to trouble BA. For example, BA profile from 10N NaOHsample showed the highest DNA peak but very low FU ([FU]=2 compared to[FU]=40). High pH seemed to trouble RT reaction. An increase of theaqueous phase pH resulted in lower CT values in Exo50 DNA Isolationwhereas EXO52 DNA isolation resulted in higher values, but there washigher DNA amount left in phenol phase compared to chloroform titration.

Decreasing pH was able to remove DNA from the EXO52 phenol phase andenrich in the aqueous phase. DNA was not harmed in the RT. RNA washarmed at highest pH. BA was affected at the three highest pH steps.

As shown in the Figures, chloroform addition was the predominant factorin determining the DNA content of the aqueous phase. A positive effectof high pH was seen only at low chloroform levels. The RNA signal wasnot affected through addition of DNA into the aqueous phase.

As shown in the Figures, there was no additive effect of pH solution toDNA copy number, and also no shift in needed chloroform amount wasnecessary to bring DNA in aqueous phase. Samples even resulted in lowercopy numbers compared to samples processed without pH solution. Onlysamples which were processed with 90 μl resulted in higher copy numbers.pH solution and higher chloroform amount did not affect RNA Isolation(mRNA). During whole titration, samples which were processed with pHsolution resulted in little lower copy numbers (except 90 μl chloroformsamples) compared to samples processed without pH solution.

As shown in the Figures, a QIAzol spin at room temperature increased thepercentage of DNA material in the aqueous phase. This was not the casewhen using higher amounts of chloroform in the EXO52 procedure.

A Qiazol centrifugation step caused DNA contamination in aqueous phase,but only in samples without PLG tube. PLG-Tube samples withcentrifugation step at room temperature also showed a little more DNA,but copy number were under LOQ=32 Copies. Temperature for centrifugationstep did not influence mRNA and miRNA isolation.

A Qiazol spin at room temperature did not add up DNA to normal EXO52 DNAisolation. There was no difference in CT values referred to the spintemperature. Temperature for centrifugation step did not influence mRNAand miRNA isolation.

As shown in the Figures, the binding and elution of DNA from EXO52 tothe RNeasy spin column did not depend on ethanol concentration in therange from 1.5× volume to 2.6× volume.

As shown in the Figures, the performance of EXO52 was not increased whenhigher ethanol concentration was used. CT values of all three assaysremained constant during whole ethanol titration. Ethanol concentrationin the pre-conditioning step of the RNA isolation did not influence therecovery of cfDNA.

As shown in the Figures, a proteinase K (ProtK) digestion of a plasmasample led specifically to loss of signal from RNA, but ProtK treatmentdid not influence DNA yield, as the same CT was obtained for allsamples.

As shown in the Figures, the DNA loading capacity of EXO52 was notreached at 8 ml plasma since the yield of DNA was still linearlyincreasing and there was no detectable DNA in the flow-through. This isin contrast to the linear loading capacity of vesicles, which is reachedat 4 mL. No cfDNA was detected in the flow through (FT) but RNA was seento accumulate from 2 mL on. The sample output is linear for DNA, but notfor RNA. RNA has a different saturation point than DNA. Adding a PLG tubto the procedure was found to increase the yield slightly. EXO52 methodadded RNA copies, when compared to commercially available CNA kits.

In some embodiments, the methods use an extraction buffer only based onguanidinium thiocyanate to extract RNA and DNA from the EXO52 column.

As shown in the Figures for RNA Isolation, 1 out of 2 replicates ofRLT+high DTT 56° C. resulted in expected CT values. Variation betweenreplicates may have been caused by clogging RNeasy membrane after addingloading mixture. BA profile showed very low RNA concentration for lefton column data point for RLT+high DTT 56° C. but only one RNA isolationresulted in expected CT values.

As shown in the Figures for DNA Isolation, AllPrep DNA column resultedin very high copy number for DNA detecting assays. Also left on columndata point showed very high CT values. DNA seemed to be lost by AllPrepDNA spin column caused by a high cut off (15-30 kb). The size of cfDNAis typically in the range of 35 bp-10 kb.

The Figures also demonstrate isolation of microRNAs using various DNA orDNA/RNA isolation procedures. The EXO52 isolated more mRNA and much moremiRNAs than the commercially available CNA kit, and EXO52 and the CNAkit isolated the same amount of DNA. The EXO52 method seemed to isolateall DNA from plasma.

As shown in the Figures, EXO52 consistently outperforms the commerciallyavailable circulating nucleic acids (CAN) kit. EXO52 has better yieldthan CNA Kit on three different plasma pools, different CNA reagentlots, different operators and different sample sources.

The EXO52 methods were used to analyze cfDNA in samples from a melanomacohort. The results obtained using the EXO52 methods were compared withthe results obtained using a commercially available CNA kit. Theintra-assay variation (based on different time points of isolation ofthe same plasma sample) of the CNA kit was higher than that observedusing the EXO52 methods. As shown in the figures, the performance of theEXO52 methods is equal or better to those obtained using thecommercially available kit.

As shown in the Figures, there was approximately 15% DNA left in organicphase after phase separation with 350 μl chloroform. Double extractionincreased DNA yield by about 15% points. Phase separation with 90 μlchloroform (RNA) followed by second extraction with additional 260 μl(sum: 350 μl chloroform) only resulted in about 50% DNA yield ascompared to normal EXO52 DNA extraction. Reloading of conditioned EXO52material onto the same column did not improve yield.

Example 2. Development of a One-Step Isolation Platform for Exosomal RNAand Cell-Free DNA from Cancer Plasma

Circulating nucleic acids in the bloodstream of cancer patients are ofgreat interest to medical research because of their potential to yieldinformation on the patient's disease status and treatment optionswithout requiring a tissue biopsy. Any diagnostic test that seeks toutilize Biofluids for mutation analysis needs a platform that canmaximize the capture of tumor derived mutations in circulation. Bloodplasma contains at least two cell-free sources of nucleic acids:circulating cell-free DNA (cfDNA), generated from apoptotic or necroticcells, and RNA enclosed in extracellular vesicles including exosomes(exoRNA), which are actively secreted by cells in the body. Since thetotal amount of nucleic acids in Biofluids is very limited and tumormutations are reflected on both RNA and DNA, a method was devised toco-isolate all exoRNA and cfDNA out of blood plasma samples into avolume small enough for effective downstream processing by RT-qPCR andtargeted re-sequencing by NGS.

FIGS. 224-226 illustrate the studies presented herein which demonstratethe following: (i) Blood plasma contains cell-free RNA in addition tocell-free DNA; (ii) EXO52 is a fast, reproducible and convenientprocedure to co-isolate all exoRNA and cfDNA from high volumes ofBiofluids; and (iii) Using both, exoRNA and cfDNA typically doubles themolecules available for rare mutant detection by qPCR and NGS.

FIGS. 227 and 228 are a series of graphs depicting the ability of theEXO52 methods provided herein to capture total circulating nucleicacids. The EXO52 methods were compared to a commercially availablecirculating nucleic acid DNA isolation kit. As shown in FIGS. 227-228,EXO52 captured all cfDNA, and EXO52 detected significantly more copiescombining exoRNA and cfDNA vs. cfDNA alone. FIG. 228 also demonstratesthat patients were identified as negative for a biomarker based solelyon cfDNA analysis, but with the combined DNA and RNA analysis, thesepatients were identified as positive for the biomarker. Those ofordinarily skill in the art will appreciate that more copies of amutation or other biomarker leads to enhanced sensitivity and accuracyin identifying mutations and other biomarkers.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following.

What is claimed is:
 1. A method for extracting cell-free DNA andmicrovesicular RNA from a biological sample comprising: (a) contactingthe biological sample with a capture surface under conditions sufficientto retain cell-free DNA and microvesicles from the biological sample onor in the capture surface, wherein the capture surface comprises amembrane that is positively charged and functionalized with quaternaryammonium; (b) contacting the capture surface with a guanidiniumthiocyanate-based extraction buffer while cell-free DNA and themicrovesicles are on or in the capture surface, thereby releasing thecell-free DNA and microvesicular RNA from the sample and producing ahomogenate; and (c) extracting the cell-free DNA, the microvesicularRNA, or both the cell-free DNA and microvesicular RNA from thehomogenate.
 2. The method of claim 1, wherein the membrane is an anionexchanger functionalized with quaternary ammonium.
 3. The method ofclaim 1, wherein the capture surface comprises three membranes that arepositively charged and functionalized with quaternary ammonium.
 4. Themethod of claim 1, wherein the biological sample is plasma, serum,urine, cerebrospinal fluid or cell culture supernatant.
 5. The method ofclaim 1, wherein the biological sample is between 0.2 to 4 mL.
 6. Themethod of claim 1, wherein step (a) further comprises processing thebiological sample by filtering the biological sample.
 7. The method ofclaim 6, wherein the filtration is performed using a 0.8 μm filter. 8.The method of claim 1, wherein step (a) further comprises acentrifugation step after contacting the biological sample with thecapture surface.
 9. The method of claim 1, wherein step (a) furthercomprises washing the capture surface after contacting the biologicalsample with the capture surface.
 10. The method of claim 1, wherein step(c) further comprises a centrifugation step after contacting the capturesurface with the guanidinium thiocyanate-based extraction buffer.
 11. Amethod for extracting cell-free DNA and microvesicular RNA from abiological sample comprising: (a) contacting the biological sample witha capture surface under conditions sufficient to retain cell-free DNAand microvesicles from the biological sample on or in the capturesurface, wherein the capture surface comprises one or more beads thatare positively charged and functionalized with quaternary ammonium; (b)contacting the capture surface with a guanidinium thiocyanate-basedextraction buffer while cell-free DNA and the microvesicles are on or inthe capture surface, thereby releasing the cell-free DNA andmicrovesicular RNA from the sample and producing a homogenate; and (c)extracting the cell-free DNA, the microvesicular RNA, or both thecell-free DNA and microvesicular RNA from the homogenate.
 12. The methodof claim 11, wherein the one or more beads are an anion exchangerfunctionalized with quaternary ammonium.
 13. The method of claim 11,wherein the biological sample is plasma, serum, urine, cerebrospinalfluid or cell culture supernatant.
 14. The method of claim 11, whereinthe biological sample is between 0.2 to 4 mL.
 15. The method of claim11, wherein step (a) further comprises processing the biological sampleby filtering the biological sample.
 16. The method of claim 15, whereinthe filtration is performed using a 0.8 μm filter.
 17. The method ofclaim 11, wherein step (a) further comprises a centrifugation step aftercontacting the biological sample with the capture surface.
 18. Themethod of claim 11, wherein step (a) further comprises washing thecapture surface after contacting the biological sample with the capturesurface.
 19. The method of claim 11, wherein step (b) further comprisesa centrifugation step after contacting the capture surface with theguanidinium thiocyanate-based extraction buffer.